Triterpene derivatives of azadirachtin having insect antifeedant and growth inhibitory activity and a process for extracting such compounds from the neem plant

ABSTRACT

The invention disclosed novel triterpene derivatives of azadirachtin of the formulae 2 to 11 of the drawings and a process for isolating new triterpene derivatives of azadirachtin from the various parts of the neem plant(Azadirachta indica A-Juss), which process comprises grinding the parts of the neem plant to get a powder, extracting the powder with a binary immiscible solvent consisting of one polar and another non-polar solvent in a ratio of 1:2 to obtain an extract, filtering the extract to get a filtrate having two layers, one layer containing lipids and the other layer containing the new triterpene derivatives of azadirachtin and water soluble constituents including sugars, separating the layers by known methods, concentrating the layer containing the new triterpenes of the formulae 2-11 including derviatives of azadirachtin and water soluble salts, treating the resultant concentrate with a polar solvent and if necessary, warm the concentrate having the solvent, and filtering/decanting the resultant solution to produce a filtrate containing the mixture of the new triterpene derivatives of azadirachtin. The lipid layer is concentrated in any known manner, the lipid concentrate is treated with a polar solvent, chilled to a temperature of 0° to -10° C. and filtered to produce a filtrate containing lipids. The new triterpene derivatives of azadirachtin are useful in suppressing the insect pest population below the economic threshold level by their antifeedant and growth inhibitory activities.

This is a divisional of application Ser. No. 07/977,618 filed on Nov.17, 1992, now U.S. Pat. No. 5,395,951.

The present invention relates to novel triterpene derivatives ofazadirachtin from neem plant (Azadirachta indica A.Juss) and a processfor preparing novel triterpene derivatives of azadirachtin. The newtriterpene derivatives of azadirachtin so prepared are useful insuppressing the insect pest population below the economic thresholdlevel by their antifeedant and growth inhibitory activities.

The imbalance in the ecosystem, human health hazards and the developmentof resistance by some insect pests caused by the continuous use ofconventional insecticides have necessitated the search for alternativesafer pesticides. Botanicals by virtue of their relatively low toxicity,biodegradability and many other factors acceptable to the environmentare considered as the best alternatives to toxic chemicals. Amongbotanicals, neem tree ranks first in view of its excellent pest controlproperties, its low mammalian toxicity and relative abundance incountries like India, Pakistan, Burma, Sri Lanka and Africa. No othertree or plant possessing insect-control properties, has received as muchattention as neem world over which is evident from the threeInternational and one National (India) conferences held during the pastdecade.

DESCRIPTION OF THE PRIOR ART

Although the protection of stored food grains, woolen clothes, fruitsand vegetables by neem leaves has been used in India since timeimmemorial, the first report of the pesticidal properties appearedaround 1927 when Mann and Burns [Agri. J. India, Calcutta 22, 325(1927)] observed during the locust cycle of 1926-27 that adult locustsdid not feed on neem leaves. This was followed by Chopra [Rept. of Dept.of Agri., Punjab Pt.2, Vol. 1, P.67 (1928)] who treated the extract ofneem leaves as contact poison on grub weevils. Since then a number ofpublications describing the various activities from different parts ofneem tree have appeared. For example, neem oil has shown antifeedantactivity against Nephotettix virescens (leaf hopper of rice). [NeemNewsletter 1 (3) 28 (1984)]. Neem seed extract has shown antifeedantactivity against Mythimna separata (rice ear cutting caterpillar) [NeemNewsletter 1 (3), 31 (1984)]. Neem cake has exhibited antifeedantactivity against Nilaparvata lugens (Rice Brown Plant Hopper) [J.Economic Entol., 77, 502 (1984)]. Neem oil has shown ovipositiondeterrent and ovicidal activity against a stored grain pest of riceCorcyra cephalonica (rice moth). Neem seed kernel water suspension hasshown oviposition deterrent activity against Spodoptera litura (Tobaccocaterpillar) [Phytoparasitica 7(3), 199 (1979)]. Neem leaves extract hasshown antifeedant activity against Rhizopertha dominica (lesser grainborer) [Neem Newsletter, 1 (2), 20 (1984)]. Neem root exudates have beenreported to contain allelochemicals.

The first active principle exhibiting antifeedant properties againstlocusts (Schistocerca gregaria) was isolated in 1968 which was named asazadirachtin by Morgan and Butterworth [Chem. Commn. 23 (1968)]. Sincethen azadirachtin has been shown to exhibit insect antifeedant and/orgrowth-inhibitory activities on more than 50 insect species. For exampleit has exhibited antifeedant activity at a dosage of 0.005% on Eariasfabia (spotted cotton ballworm, family, Lepidoptera) [Phytoparasitica, 9(1), 27 (1981)]. It has been shown growth inhibitory activity at adosage of 5-10 ppm against Spodoptera litura (tobacco caterpillar,Lepidoptera [Proceedings of the National Seminar on Neem in Agri., ]IARI, New Delhi, (1983); Indian J. Expt. Biol. 23(3), 16 (1985) and LD₅₀of azadirachtin against S. litura is reported to be 1.1 g/g [J. Entomol.Res., 11 (2), 166 (1987)]. It has been reported to exhibit sterilant,insecticidal, delay in egg hatching, growth inhibitory and antifeedantactivities at a dosage of 10-100 ppm against Epilachna verivestis(Mexican bean bettle, Coleoptera [Z pflakrankh pflaschutz, 82 (3), 176(1975); Z. Angew Entomol. 93, 12 (1982);

Systemic action of azadirachtin through roots to leaves has beenreported by Saxena et al [J. Econ. Entomol., 77 (2) 502 (1984)].

The drawing accompanying this specification shows the structure ofazadirachtin; in formula 1 which was established in 1986 by three groupsof workers [Tetrahedron, b 43, 2789; 2805, 2817 (1987)].

Till this time more than 80 tetranortriterpenes and derivatives ofazadiraachtin have been isolated from neem extracts. Some of them arementioned here: -nimbinene, nimbandiol, azadiradione, salannin,vilasinin, gedunin, meldenindiol and nimbin. Out of them at least 35triterpene derivatives show either insect antifeedant activity, orgrowth-inhibitory activity or both. For example azadirachtin A, B, D, E,F and G show insect growth inhibitory activities against Epilachnaverivestis at a dosage of 1-10 ppm [Insecticides of Plant Origin, ACSSymposium series, 387, 150 (1989(]. Similarly 3-tigloylazadirachtol hasexhibited 97% antifeedant activity at a dosage of 1.0 ppm [Tetrahedron,45, 5175 (1989)]. Amongst other compounds azadiradione, azadirone,6-deacetylnimbinene, epoxyazadiradione, gedunin, nimbandiol, salanninand salannol have shown antifeedant and growth-inhibitory activityagainst E. Verivestis [Proceedings, 2nd International Neem Conference181 (1983)]. Azadirachtin has been reported to be active againstnematodes and whitegurbs [Entomol. Exp. Appl., 24, 448 (1978).

Synthetic insecticides belonging to the class of organochlorine andorganophosphorous are found to be quite toxic to mammals, fish,honeybees etc. They pollute the ecosystem by their toxicity and residualaction. In addition they cause serious health hazards to human beings.In view of this there is an urgent need to look for safer alternativesand plant products appear to be the right choice because of theirrelatively low toxicity and biodegradability.

With the above objective we have made extensive research on the pestcontrol properties of the extract from neem seeds and have observed thatif the underside components viz. the saturated fats and the watersoluble compounds including sugars are removed, the resulting extract isenriched with the required active compounds viz. the triterpenoids andthe unsaturated fats.

Neem seed extract contains essentially four components namely saturatedand unsaturated fats, triterpenoids and water soluble compoundscontaining sugars. Of these, triterpenoids exhibit insect-antifeedantand growth-inhibitory activity while the unsaturated fats possessinsecticidal activity against aphids, mites, jassids, cotton white fliesand other soft bodies insects. Of the remaining two, saturated fats areinactive and phytotoxic (above 2% dosage) and the water soluble sugarsact as insect attractants and fungal growth promoters. Both these areundesirable in the formulation of pest control agents from neem.

In the known processes of preparing insecticidal composition from neemno attempt has been made to separate all the above said four componentsso that the concentration of the required compounds, the triterpenoidsand the unsaturated fats can be used advantageously for pest controlpurposes. Some of the processes involve expelling neem oil whichcontains a low percentage (0.05) of active triterpenoids and directlyuse for pest control purposes. In some other processes the neem isextracted using a solvent like (hexane) to obtain an oil containing lowpercentage (0.05) of the active triterpenoids. Consequently, due to thevery low percentage of the terpenoids these oils used directly for pestcontrol purposes do not have desirable pest control properties.

A mixture of neem oil, karanja oil, mohua oil, gingely oil, castor oilhas also been used but it exhibits phytotoxic properties in fieldtrials. Neem based fertilizer in which neem extractive (crude) isblended with urea and sold as a fertilizer is also commerciallyavailable.

In the process of the present invention we have removed all thedrawbacks of the existing processes by complete extraction of neem seedpowder and solvent partitioning in one step using two immisciblesolvents, forming two layers, one solvent layer containing the lipidsand the other solvent layer containing the triterpenes and water solublecompounds including sugars. The second solvent layer is treated with apolar solvent in which the triterpenes only are soluble, thus separatingthem from the undersired sugar fraction by filtration/decantation. Thus,in two steps the active rich triterpenoid fraction exhibiting insectantifeedant and growth-inhibitory activity is obtained which is freefrom the undesirable lipids and water soluble compounds includingsugars. The percentage of the active triterpenoid fraction is about 2.5%based on the seed powder.

Similarly the separation of the undesirable saturated fat (9%) from thetotal lipids has been achieved by fractional crystallisation fromacetone at 0°-10° C. The saturated fats, at the above temperaturesolidify and are removed by filtration and the unsaturated fats remainin the mother liquor.

During our continued research in the field of pest control agents weobserved that 1) neem seed extract is more active biologically than theextract of any other part of the neem tree. 2) Neem seed extract is moreactive than neem seed kernel extract as the husk or hull of the seedsalso contains active principles. 3) Neem seed extract is more activethan the expelled oil. 4) Triterpene fraction of the seed extractexhibits antifeedant activity and insect-growth inhibiting activity,while the lipid fraction rich in unsaturated fats is responsible foraphidicidal, miticidal and insecticidal activity against sucking pestsand soft bodied insects.

SUMMARY OF THE INVENTION

The present invention provides new triterpene derivatives ofazardirchtin from the parts of neem plant (A indica). These newtriterpenes have the formulas 2-11 shown in the drawings accompanyingthis specification.

Another object of the present invention is to provide a process for thepreparation of the new triterpenes.

Yet another object of the present invention is to provide a process forthe preparation of an extract containing these new triterpenes which areuseful as insecticidal compositions.

Still another object of the present invention is to provide a processfor the preparation of insecticidal compositions containing lipids.

Though any part of the neem plant can be used in carrying out thepresent invention disclosed herein, it is preferred to employ the seedor the neem cake powder due to the above findings that neem seed extractand neem seed cake extract have excellent insect-control activity.

DRAWINGS

FIG. 1--shows the structure of azadirachtin.

FIG. 2--shows triterpenes of Formulas 2-4.

FIG. 3--shows triterpenes of Formulas 5-8.

FIG. 4--shows triterpenes of Formulas 9-11.

FIG. 5--is the HPLC chromatogram of the triperpene active fraction.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, present invention provides a process for the isolation ofnew triterpene derivatives of azadirachtin having the formulas 2-11shown in the drawing accompanying the specification, from the parts ofthe neem plant, which comprises:

a. grinding the parts of the neem plant to get a powder;

b. extracting the powder with a binary immiscible solvent consisting ofone polar and another non-polar solvent in the ratio of 1:2 to obtain anextract;

c. filtering the extract to obtain a filtrate having 2 layers--one layercontaining lipids and another layer containing new triterpenes,azadirachtin and water soluble sugars;

d. separating the layers by known methods;

e. concentrating the polar solvent layer containing the new triterpenederivatives of azadirachtin and water soluble constituents

f. treating the resultant concentrate with a polar solvent; and

g. filtering/decanting the resultant solution to produce a filtratecontaining the mixture of new triterpene derivatives of azadirachtin ofthe Formulas 2-11 and known triterpenes including azadirachtin,separating the new triterpene derivatives of azadirachtin by columnchromatography.

The separation of the new derivatives may be done using elutes such asethyl acetate, benzene, acetone, and pet-ether. 120 g oftriterpene-active fraction was subjected to column chromatography overSilica gel (60-120 mesh size, 2 kg) using acetone:pet-ether as theelution gradient to collect four broad fractions A (25 g), B (20 g), C(45 g) and D (20 g).

Fraction D (20 g) was put for further fractionation by columnchromatography over Silica gel (60-120 mesh, 750 g) usingchloroform:methylcyanide as the elution gradient to collect five broadfractions D1 (4 g), D2 (5 g), D3 (1.5 g), D4 (2 g), D5 (7 g) whichcontained mainly the azadirachtin and azadirachtin derivatives. Out ofthese, D1 and D2 fractions contained mainly salannin, salanno,nimbandiol and vilasinnin and its derivatives. D3 and D4 fractionscontained azadirachtin and its derivatives and D5 fraction containedmostly polar compounds.

Azadirachtin and its new derivatives were isolated from D3 and D4fractions by repeated column chromatography and preparative TLC usingdifferent solvent mixtures such as acetone:pet-ether (4:6),ethylacetate:benzene (9:1) and chloroform:methylcyanide (5:2). The Rfvalues of the ten new azadirachtin derivatives are different in eachsolvent system. But we could say that the compound No. (2), (4), (8),(9) and (10) are less polar than azadirachtin in all of the three abovesolvent systems and the remaining five compounds are more polar thanazadirachtin. The structures of the above new azadiirachtin derivativeswere established by spectral data (Chart 1 & 2).

    __________________________________________________________________________    .sup.1 H-NMR DATA OF COMPOUND 2-11 CHART-1)                                   __________________________________________________________________________    Serial No.                                                                            2      3      4      5      6                                         __________________________________________________________________________    1-H     3.58(m)                                                                              4.68(dd)                                                                             4.66(dd)                                                                             3.40(m)                                                                              5.34(dd)                                                 (2.75;3.0)                                                                           (2.7;3.1)     (2.65;2.60)                               2-H     2.32(ddd)                                                                            2.36(ddd)                                                                            2.34(ddd)                                                                            2.38(ddd)                                                                            2.32(ddd)                                         (16.2;2.65;3.1)                                                                      (16.3:2.8;3.2)                                                                       (16.2;2.8;3.1)                                                                       (16.3;2.65;3)                                                                        (16.38;3.48;3.22)                         2-H     2.12(ddd)                                                                            2.16(ddd)                                                                            2.20(ddd)                                                                            2.32(ddd)                                                                            2.29(ddd)                                         (16.2;2.95;2.8)                                                                      (16.3;3.0;2.8)                                                                       (16.2;3.0;2.8)                                                                       (16.3;3.1;2.65)                                                                      (16.38;3.22;3.11)                         3-H     5.45(t)                                                                              4.48(t)                                                                              5.44(t)                                                                              5.48(t)                                                                              5.50(t)                                           (2.7;3.1)                                                                            (2.7;2.8)                                                                            (2.7;2.9)                                                                            (2.75;2.8)                                                                           (2.7;3.08)                                5-H     3.3(d) 3.09(d)                                                                              3.30(d)                                                                              3.24(d)                                                                              3.34(d)                                           (12.5) (12.5) (12.5) (12.5) (12.5)                                    6-H     4.40(dd)                                                                             4.15(dd)                                                                             4.44(dd)                                                                             4.48(dd)                                                                             4.43(dd)                                          (12.5;2.7)                                                                           (12.5;2.75)                                                                          (12.5;2.8)                                                                           (12.5;2.65)                                                                          (12.5;3.73)                               7-H     4.62(d)                                                                              4.65(d)                                                                              4.64(d)                                                                              4.8(d) 4.62(d)                                           (2.7)  (2.65) (2.65) (2.63) (2.2)                                     9-H     2.62(s)                                                                              3.32(s)                                                                              3.30(s)                                                                              3.30(s)                                                                              2.62(s)                                   11-H    4.50(s)                                                                              --     --     4.53(s)                                                                              5.38(s)                                   15-H    4.56(d)                                                                              4.58(d)                                                                              4.64(d)                                                                              4.50(d)                                                                              4.55(d)                                           (3.20) (3.20) (3.20) (3.20) (3.20)                                    16-H    1.65(ddd)                                                                            1.60(ddd)                                                                            1.65(ddd)                                                                            1.70(ddd)                                                                            1.66(ddd)                                         (13.0;3.5;5.3)                                                                       (13.0;3.5;5.3)                                                                       (13.0;3.5;5.4)                                                                       (13.0;3.35;5.0)                                                                      (13.0;3.8;5.8)                            16-H    1.28(d)                                                                              1.32(d)                                                                              1.30(d)                                                                              1.32(d)                                                                              1.25(d)                                           (13.2) (13.0) (13.0) (13.0) (13.27)                                   17-H    2.40   2.38(d)                                                                              2.36(d)                                                                              2.40(d)                                                                              2.34(d)                                           (5.0)  (5.0)  (5.0)  (5.0)  (5.03)                                    18-H    1.95(s)                                                                              2.05(s)                                                                              1.198(s)                                                                             2.08(s)                                                                              1.93(s)                                   19-H    3.58(d)                                                                              3.62(s)                                                                              3.60(d)                                                                              3.62(d)                                                                              3.77(s)                                           (9.6)  (8.0)  (8.5)  (9.2)                                            19-H    4.00(d)                                                                              3.62(s)                                                                              4.02(d)                                                                              3.90(d)                                                                              3.77(s)                                           (9.6)  (8.0)  (8.5)  (9.2)                                            21-H    5.65(s)                                                                              5.64(s)                                                                              5.60(s)                                                                              5.60(s)                                                                              5.63(s)                                   22-H    5.05(d)                                                                              5.00(d)                                                                              5.0(d) 5.06(d)                                                                              5.03(d)                                           (2.9)  (2.9)  (2.9)  (2.85) (2.9)                                     23-H    6.40(d)                                                                              6.42(d)                                                                              6.42(d)                                                                              6.44(d)                                                                              6.48(d)                                           (2.9)  (2.9)  (2.9)  (2.85) (2.9)                                     28-H    4.02(d)                                                                              3.80(d)                                                                              4.16(d)                                                                              4.06(d)                                                                              4.06(d)                                           (9.0)  (7.6)  (7.8)  (9.0)  (8.76)                                    28-H    3.80(d)                                                                              3.70(d)                                                                              3.80(d)                                                                              3.74(d)                                                                              3.72(d)                                           (9.0)  (7.6)  (7.8)  (9.0)  (8.8)                                     29-H    --     1.04(s)                                                                              --     --     --                                        30-H    1.76(s)                                                                              1.74(s)                                                                              1.73(s)                                                                              1.42(s)                                                                              1.29(s)                                   7-CH    --     2.78(brs)                                                                            --     --     2.8(brs)                                  11-CH   --     5.00(s)                                                                              --     --     3.26(br.s)                                14-CH   --     3.06(br.s)                                                                           --     --     3.06(br.s)                                12-OCH.sub.3                                                                          3.65(s)                                                                              --     3.65(s)                                                                              3.68(s)                                                                              --                                        29-OCH.sub.3                                                                          3.76(s)                                                                              3.77 (s)                                                                             3.75 (s)                                                                             3.78(s)                                                                              3.77(s)                                   CH.sub.3 COO                                                                          1.84(s)                                                                              1.83(s)                                                                              1.93(s)                                                                              2.02(s)                                                                              1.83(s)                                   Tigloyl 6.88(qq)                                                                             6.95(qq)                                                                             Benzoyl gr.                                                                          --     6.95(qq)                                  3'-H    (7.0;1.3)                                                                            (7.0;1.3)                                                                            2'-7.9(m)                                                                            --     (7.0;1.34)                                                      6'-7.7(m)                                               4'-H    1.76(dq)                                                                             1.74(dq)                                                                             3'-7.57(m)                                                                           --     1.74(dq)                                          (7.0;1.01)                                                                           (7.0;1.01)                                                                           4'-7.4(m)                                                                            --     (7.06,1.01)                                                     5'-7.6(m)                                               5'-H    1.84(dq)                                                                             1.81(dq)                                                                             --     --     1.81(dq)                                          1.5,1.01)                                                                            (1.5;1.01)           (1.5;1.01)                                __________________________________________________________________________    Compound No.                                                                          7      8      9      10     11                                        __________________________________________________________________________    1-H     4.93(t)                                                                              4.60(t)                                                                              3.74(t)                                                                              4.72(t)                                                                              3.62(dd)                                          (2.9;3.1)                                                                            (2.85;3)                                                                             (2.9;3.0)                                                                            (2.9;3.1)                                                                            (2.9;3.0)                                 2-H     2.38(ddd)                                                                            2.33(ddd)                                                                            2.36(ddd)                                                                            2.28(ddd)                                                                            2.76(ddd)                                         (16.0;2.9;2.7)                                                                       (16.0;2.7;2.8)                                                                       (16.0;2.6;2.8)                                                                       (16.2,2.9,2.6)                                                                       (16.2;2.9;2.65)                           2-H     2.14(ddd)                                                                            2.14(ddd)                                                                            2.16(ddd)                                                                            2.13(ddd)                                                                            2.12(ddd)                                         (16.0;2.7;2.8)                                                                       (16.0;2.7;2.8)                                                                       (16.0;2.6;2.8)                                                                       (16.2;2.6;2.9)                                                                       (16.2;2.6;2.85)                           3-H     5.30(t)                                                                              5.15(dd)                                                                             5.45(t)                                                                              5.48(t)                                                                              3.76(t)                                           (2.9;3.1)                                                                            (2.9;3.1)                                                                            (2.9;3.0)                                                                            (2.9;3.0)                                                                            (2.8;3.2)                                 5-H     3.10(d)                                                                              3.30(d)                                                                              3.30(d)                                                                              3.30(d)                                                                              3.16(d)                                           (12.5) (12.5) (12.5) (12.5) (12.5)                                    6-H     4.04(dd)                                                                             4.04(dd)                                                                             4.13(dd)                                                                             4.42(dd)                                                                             4.16(d)                                           (12.5;3)                                                                             (12.5;3.0)                                                                           (12.5;3)                                                                             (12.5;3.2)                                                                           (12.5;3.0)                                7-H     4.65(s)                                                                              4.44(d)                                                                              4.64(d)                                                                              4.48(d)                                                                              4.62(d)                                           (3.0)  (3.0)  (3.20) (3.2)  (3.0)                                     9-H     2.60(s)                                                                              4.24(s)                                                                              4.02(s)                                                                              3.48(S)                                                                              4.02(s)                                   11-H    3.75(m)                                                                              --     --     --     --                                                --     3.85(d)(14.0)                                                                        --     --     --                                        12-H    3.92(br.s)                                                                           5.40(d)(14.0)                                                                        --     --     --                                        15-H    4.55(d)                                                                              4.62(d)                                                                              4.2(d) 4.18(m)                                                                              4.60(d)                                           (3.2)  (3.2)  (3.0)         (3.0)                                     16-H    1.60(ddd)                                                                            1.60(ddd)                                                                            1.67(ddd)                                                                            1.80(m)                                                                              1.65(m)                                           (13.0;.3.4;5.0)                                                                      (13.0;3.4;5.0)                                                                       (13.0;3.4;5.1)                                          16-H    1.26(ddd)                                                                            1.26(d)                                                                              1.30(d)                                                                              1.90(m)                                                                              1.92(m)                                           (13.0) (5.0)  (5.0)                                                   17-H    2.40(d)                                                                              2.58(d)                                                                              2.42(d)                                                                              2.16(m)                                                                              2.14(m)                                           (5.0)  (5.0)  (5.0)                                                   18-H    2.00(s)                                                                              1.98(s)                                                                              2.07(s)                                                                              1.55(s)                                                                              2.04(s)                                   19-H    CH.sub.3 -1.32(s)                                                                    1.23(s)                                                                              1.40(s)                                                                              3.66(d)                                                                              CH.sub.3 -1.45(s)                                                      (9.7)                                            19-H                         4.28(d)                                                                       (9.7)                                            21-H    5.62(s)                                                                              5.60(s)                                                                              5.62(s)                                                                              5.65(s)                                                                              5.62(s)                                   22-H    5.05(d)                                                                              5.05(s)                                                                              5.04(s)                                                                              4.88(s)                                                                              1.88(s)                                           (2.90) (2.90) (3.0)  (2.9)  (2.9)                                     23-H    6.40(d)                                                                              6.40(d)                                                                              6.40(d)                                                                              6.36(d)                                                                              6.36(d)                                           (2.90) (2.90) (3.0)  (2.9)  (2.9)                                     28-H    3.70(br.s)                                                                           4.55(s)                                                                              3.72(d)                                                                              3.69(d)                                                                              3.65(d)                                                  (9.0)  (9.6)  (8.8).sub.--                                                                         (8.6)                                     28-H    3.70(br.s)                                                                           3.85(d)                                                                              3.60(d)                                                                              4.04(d)                                                                              3.92(d)                                                  (9.0)  (9.0)  (8.8)  (8.5)                                     29-H    1.05(s)                                                                              --     --     --     --                                        30-H    1.58(s)                                                                              1.68(s)                                                                              1.73(s)                                                                              1.68(s)                                                                              1.60(s)                                   7-CH    --     2.58(br.s)                                                                           2.8(br.s)                                                                            --     --                                        11-CH   --     --     --     --     --                                        14-CH   --     2.92(br.s)                                                                           2.95(br.s)                                                                           --     --                                        12-OCH.sub.3                                                                          --     --     --     3.68(s)                                                                              3.68(s)                                   29-OCH.sub.3                                                                          --     3.72(s)                                                                              3.78(s)                                                                              3.76(s)                                                                              3.78(s)                                                  1.98(s)                                                        CH.sub.3 COO                                                                          1.86(s)                                                                              1.92(s)                                                                              2.02(s)                                                                              1.98(s)                                                                              --                                        Tigloyl 6.93(qq)                                                                             6.94(qq)                                                                             --     6.88(qq)                                         3'-H    (7.0;1.34)                                                                           (7.0;1.30)                                                                           --     (7.0;1.3)                                        4'-H    1.78(dq)                                                                             1.78(dq)                                                                             --     1.75(dq)                                                 (7.0;1.1)                                                                            (7.0;1.1)                                                                            --     (7.0;1.1)                                                                            --                                        5'-H    1.83(dq)                                                                             1.84(dq)                                                                             --     1.84(dq)                                                 (1.34;1.1)                                                                           (1.30;1.1)                                                                           --     (1.3;1.11)                                       __________________________________________________________________________

                  CHART-2                                                         ______________________________________                                        .sup.13 C-NMR DATA COMPOUND 3 and 6                                                        3          6                                                     ______________________________________                                        C-1            70.8478(d)   72.928(d)                                         C-2            29.5797(t)   29.577(t)                                         C-3            68.9088(d)   67.115(d)                                         C-4            42.3927(s)   52.3129(s)                                        C-5            35.663(d)    36.998(d)                                         C-6            74.9161(d)   72.215(d)                                         C-7            76.4206(d)   74.018(d)                                         C-8            44.9545(s)   43.335(s)                                         C-9            44.7298(d)   48.499(d)                                         C-10           49.8957(s)   47.760(s)                                         C-11           103.9192(s)  100.911(d)                                        C-12           171.6262(s)  --                                                C-13           69.9268(s)   66.917(s)                                         C-14           69.9220(s)   69.946(s)                                         C-15           76.2910(d)   76.147(d)                                         C-16           24.9646(t)   25.255(t)                                         C-17           48.8888(d)   48.132(d)                                         C-18           18.2318(q)   18.570(q)                                         C-19           72.5881(t)   72.928(t)                                         C-20           83.4946(s)   83.384(s)                                         C-21           108.8309(d)  108.545(d)                                        C-22           107.2595(d)  107.509(d)                                        C-23           146.8451(d)  147.074(d)                                        C-28           77.35(t)     77.425(t)                                         C-29           18.9414(q)   173.428(s)                                        C-30           21.0782(q)   20.824(q)                                         COOCH.sub.3    53.0748(q)   52.452(q)                                         CH.sub.3 COO   169.7945(s)  169.657(s)                                        CH.sub.3 COO   20.7701(q)   2.485(q)                                          OTig;                                                                         C-1'           166.1988(s)  166.510(s)                                        C-2'           128.6384(s)  128.689(s)                                        C-3'           137,1983(d)  137.941(d)                                        C-4'           14.1673(q)   14.237(q)                                         C-5'           11.8579(q)   11.804(q)                                         ______________________________________                                    

According to a feature of the invention, there is provided a process forthe preparation of new insecticidally active compositions containing theabove referred new triterpenes including azadirachtin and itsderivatives from the neem plant which process comprises:

a. grinding the parts of the neem plants to get a powder;

b. extracting the powder with a binary immiscible solvents consisting ofone polar and the other non-polar solvent in the ratio of 1:2 to obtainan extract;

c. filtering the resultant extract to get a filtrate having two layersone layer containing lipids, and other containing the new triterpenesincluding azadirachtin and its derivatives and water solubleconstituents including sugars;

d. separating the layers by known methods;

e. concentrating the layer containing triterpenes including theazadirachtin and its derivatives and water soluble constituentsincluding sugars;

f. treating the resultant concentrate with a polar solvent and

g. filtering the resultant solution to produce a filtrate containing themixture of new triterpenes including azadirachtin and its derivatives.

It is found that the fraction in the polar solvent phase consists oftriterpenes, azadirachtin and its derivatives in the range of 40-60%alongwith some water soluble compounds such as sugars, glucosides, andamino acids. The non-terpenenic portion which has been found to be watersoluble can easily be removed from the terpenic portion by any knownmethods such as by partitioning between water and water immiscible polarsolvents. Examples of water immiscible polar solvents which can be usedare ethylenedichloride, chloroform, carbontetrachloride, ethylacetate,n-butanol, isobutanol and the like. Alternatively, the triterpenicactive fraction can be separated by dissolving it in polar solvents likeethylenedichloride, ethylacetate, acetone and chloroform. The finaltriterpene fraction obtained by this method amounts to 2-5% based on theseed powder. This fraction contains at least 35 triterpenes related toazadirachtin of which 25-30 are biologically active. The knowntriterpenes include salannin, gedunin, azadiradione, azadirone, salannolacetate, epoxyazadiradione, nimbandiol, salannin derivatives, salannolderivatives, azadirchtin and new derivatives of azadirachtin. All thesecompounds are found to exhibit insect antifeedant and growth inhibitoryactivities.

The lipid fraction (i.e. the layer containing the lipids) obtainedduring partitioning in the non-polar solvent i.e. step c) consists ofmono-, di- and triglycerides of fatty acids, sulphur compounds, straightchain hydrocarbons, straight chain esters of fatty alcohols and acids,phyosterols and their esters and less polar triterpenes and free fattyacids of the type, oleic, stearic, linoleic, arachidoic and tiglic isfound to exhibit insecticidal activity against soft bodied insects andsucking pests such as aphids, mites, cotton white fly, jassids, etc. Theactivity of the fraction is enhanced by dissolving the total lipidfraction in a polar solvent and chilling the resultant solution to atemperature in the range of 0°-10° C. for a period ranging from 10-20hrs. The solid separating out under the above conditions is filtered andthe solvent removed from the filtrate preferably by distillation to givethe active fraction. This active fraction consisting of enrichedunsaturated lipids is found to constitute 8-12% based on the seeds andin the case of neem cake it is found to be 4-6%.

The liquid fraction (2 kg) was dissolved in acetone (3 liters) and thesolution was chilled at 0° to -10° C. for 12 hrs. The chilled productwas filtered quickly and the filtrate was concentrated by distillingacetone and the residue obtained weighed 1 kg. It consists of mostlyunsaturated fatty acids, unsaturated glycerides, hydrocarbons, sulphurcompounds, phytosterols (sitosterol, stigmsterol, campesterol) and lesspolar triterpenes such as gedunin, azadirone, zadirodione, nimbin,deacetylnmibin etc.

According to another embodiment of the present invention there isprovided a process for the preparation of a new insecticidally activecomposition containing lipids enriched with unsaturated fats from thepart of the neem plant which comprises:

a. grinding the parts of the neem plant to get a powder;

b. extracting the powder with a mixture of two immiscible solventsconsisting of one polar and other non-polar in the ratio of 1:2 toobtain an extract;

c. filtering the resultant extract to get a filtrate having twoseparable layers, one layer containing the lipids and the othercontaining the triterpenes including azadirachtin and the derivatives ofazadirachtin and water soluble constituents including sugars;

d. separating the layers by known methods;

e. concentrating the non-polar solvent layer containing the lipids toobtain the total lipids and

f. dissolving the above lipids in a polar solvent and chilling thesolution in the range of 0° to -10° C. for 12 hrs and filtering toproduce a filtrate containing unsaturated lipids.

By way of examples the polar solvents which can be employed arechloroform, ethanol, methanol, isobutanol, n-butanol, ethylenedichlorideand the like. The non-polar solvents which can be used are selected fromhexane, heptane, pentane, benzene, toluene and the like.

The present invention is illustrated by the examples given below whichshould not be constructed to limit the scope of the invention.

EXAMPLE 1

Neem seeds which were stored for at least for 2-4 months aftercollection were produced and 10 kg of the powder was stirred with 30liters of methanol:hexane (1:2) for 2 hours and filtered. The filtratecontaining two layers, one of methanol and the other of hexane wereseparated and the solvents removed from both the layers to yield thetriterpenes alongwith water soluble polar compounds 0.7 kg. The lipidportion weighed 1.8 kg. 0.7 kg of the methanol extract was treated withwarm (about 40° C.) acetone and decanted. The clear solution wasstripped off the solvent to give triterpene-rich insect control agents,250 g.

The triterpene active fraction (120 g) was separated by using columngrade Silica gel (60-120 mesh size) and acetone:pet-ether solventmixture with increasing percentage of acetone as the elution gradientwas used to collect four broad fractions A (25 g), B (32 g), C (45 g)and D (18 g).

Rechromatography of fraction D (18 g) on Silica gel using ethylacetate-benzene with increasing polarity of ethyl acetate resulted infive broad fractions D1 (4 g), D2 (6 g), D3 (1.5 g), D4 (2 g) and D5(4.5 g). D3 (1.5 g) fraction was again rechromatographed on Silica gel(60-120 mesh) with chloroform:methylcyanide as the eluent with theincreasing percentage of methylcyanide to give the mixture of compounds(2), (4), (8), (9) and (10) along with less polar and more polarcompounds. Preparative TLC on Silica gel (400 mesh) of this fractionwith ethylacetate benzene (9:1) enriched the above (2), (4), (8), (9)and (10) compounds. The enriched fraction containing the above compoundswas separated by preparative TLC Silica gel (400 mesh size) withchloroform:methylcyandie (5:2) as the solvent system gave each compound75% purity. Again each compound was purified by preparative TLC (Silicagel, 400 mesh) with acetone:pet-ether (4:6) as solvent system gave thepure compounds (2), (4), (8), (9) and (10).

D₄ (2 g) fraction was again subjected to column chromatography overcolumn grade Silica gel (60-120 mesh) using ethylacetate:benzene withincreasing percentage of ethylacetate which gave a crude mixture of fivemore polar compounds (3) (5), (6), (7) and (11) along with the less andmore polar minor components. The above crude mixture was put onpreparative TLC (Silica gel, 400 mesh size) withchloroform:methylcyanide (5:2) which gave each compound in 50% pureform. Each of the above 50% pure compounds was again loaded onpreparative TLC (Silica gel, 400 mesh size) with acetone:pet-ether (4:6)to enrich it to 90% purity and again each was finally purified to obtainall the more polar compounds (3), (5), (6), (7) and (11) by usingethylacetate:benzene (9:1) as solvent system on preparative TLC (400mesh size, Silica gel).

EXAMPLE 2

Neem seeds which were stored atleast for 2-4 months after collectionwere powdered and 10 kg of the powder was stirred with 30 liters ofmethanol:hexane (1:2) for 2 hours and filtered. The filtrate containingtwo layers, one of methanol and other of hexane were separated and thesolvents removed from both the layers to yield the triterpenes alongwithwater soluble polar compounds 0.7 kg. The lipid portion weighed 1.8 kg.0.7 kg of methanol extract was treated with warm (about 40° C.) acetoneand decanted. The clear solution was stripped off the solvent to givetriterpene-rich pest control agents 250 g.

The triterpene-active fraction (150 g) was separated by using columngrade Silica gel (60-120 mesh size) and acetone:pet-ether solvent systemwith increasing percentage of acetone as the elution gradient to collectfour broad fractions A (30 g), B (42 g), C (55 g) and D (26 g).

Rechromatography of fraction D (26 g) on Silica gel (60-120 mesh) usingchloroform:methylcyanide resulted in five broad fractions D₁ (9 g), D₂(4 g), D₃ (4 g), D₄ (5 g) and D₅ (4 g).

D₃ (4 g) fraction was again put on column for further separation overSilica gel (60-120 mesh size) with acetone:pet-ether with increasingpercentage of acetone which gave the mixture of (2), (4), (8), (9) and(10) alongwith less and more polar constituents. Preparative TLC of thismixture on Silica gel (400 mesh size) enriched the above five compounds(2), (4), (8), (9) and (10) with ethylacetate:benzene (9:1) to 50%purity. The enriched fraction containing the above compounds was furtherseparated by preparative TLC on Silica get (400 mesh size) withchloroform:methylcyanide as solvent system (5:2) to give each of theabove compounds in 75% purity. Again each one was purified onpreparative TLC (Silica gel, 400 mesh size) with acetone:pet-ether (4:6)which gave the pure compounds.

D₄ (4 g) fraction was again subjected to column chromatography overcolumn grade Silica gel (60-120 mesh) using chloroform:methylcyanidewith increasing percentage of methylcyanide which gave a crude mixtureof five more polar compounds (3), (5), (6), (7) and (11) alongwith theless and more polar compounds. The above crude mixture was separated onpreparative TLC (Silica gel, 400 mesh size) with ethylacetate:benzene(9:1) which gave each compounds in 50% pure form. The above 50% wasagain loaded on preparative TLC (Silica gel, 400 mesh size) withchloroform:methylcyanide (5:2) to give 90% pure compounds and finallythey were purified by preparative TLC using acetone:pet-ether (4:6) toget very pure compounds viz. (3), (5), (6), (7) and (11).

The antifeedant activity of all the compounds was evaluated as follows:

A test solution containing 20 ppm of each compound was prepared. Weigheddiscs of leaves were treated with the above solution and the larvae ofHeliothis armigera were released on these treated leaves. An untreatedcontrol experiment was run simultaneously. The weight of the leavesafter 24 hrs and 48 hrs were recorded to evaluate the antifeedantactivity of the compounds. Table 1 shows antifeedant activity ofcompounds 3,5 and 6.

                  TABLE 1                                                         ______________________________________                                        Compound of    % Protection                                                   the formula    After 24 hr                                                                             After 48 hr                                          ______________________________________                                        3              50        60                                                   5              50        60                                                   6              75        80                                                   ______________________________________                                    

EXAMPLE 3

Neem seeds which were stored for at least 2-4 months after collection,were powdered and 10 kg of the powder was stirred with 30 liters ofmethanol:hexane (1:2) for 2 hrs and filtered. The filtrate containingtwo layers, one of the methanol and the other of hexane were separatedby using a separatory funnel and the solvents removed from both thelayers to yield triterpenes including azadirachtin and its derivativesalong with water soluble polar compounds weighing 0.7 kg. The lipidportion weighed 1.8 kg.

0.7 kg of the aqueous methanol extract was treated with 7 liters of warmacetone (35°-40° C.) in three lots and the acetone solubles weredecanted and the solvent removed to yield the triterpene active fractionweighing 250 gms. The residue (450 gms) which was insoluble in acetonewas discarded. Azadirachtin content in the above triterepene activemixture was estimated by HPLC using (60:40) methanol:water 1 ml/ml C-18reverse phase column and 215 nm UV detector. Azadirachtin peak wasidentified by comparing with that of authentic sample and the percentageof azadirchatin was found to be 8%.

EXAMPLE 4

Neem cake (10 kg) obtained by expelling the oil in an expeller wasstirred with 90% aq. methanol and hexane (30 liters, 1:2) for 2 hrs andfiltered. The aqueous methanol layer and the hexane layer were separatedin a separatory funnel and the solvents removed separately to give 0.8kg of the aqueous methanol solubles and 0.8 kg of lipid fractionrespectively.

The aqueous methanol solubles 0.8 kg was shaken with 10 liters ofisobutanol and water (1:1) and isobutanol layer was separated in aseparatory funnel which was distilled off to give triterpene activefraction, 280 gms.

Azadirachtin content of the above triterpene active fraction using HPLC(50:50, methanol:water, 1 ml/ml C-18 reverse phase column) and the UVdetector at 217 nm was found to be 10%.

EXAMPLE 5

Neem seed powder (10 kg) was extracted with stirring with a mixture ofaqueous methanol (85%) and heptane (30 liters, 1:2) and filtered. Thefiltrate consisting of both aqueous methanol and heptane layers wasseparated in a separatory funnel and the solvents removed to giveaqueous methanol solubles (0.75 kg) and the lipid fraction in theheptane solubles 2.0 kg).

Methanol solubles (0.75 kg) were partitioned between ethylenedichlorideand water (3 liters each) and the ethylenedichloride layer was separatedand concentrated to give 250 gms of triterpene active fraction.Azadirachtin content using HPLC at 215 nm UV wavelength andacetonitrile:water (60:40) 1 ml/ml C-18 reverse phase column was foundto be 9%.

1. The HPLC chromatogram of the triterpene active fraction is depictedin FIG. 5.

2. The bioefficacy data generated for triterpene active fraction at 1000ppm are given in Table 2.

                  TABLE 2                                                         ______________________________________                                        BIOEFFICACY DATA OF TRITERPENE ACTIVE                                         FRACTION DURING FIELD TRIALS ON VARIOUS                                       CROPS AND COMPARISON WITH OTHER NEEM                                          BASED INSECT-CONTROL AGENTS.                                                  ______________________________________                                        Tobacco nursery    Plot size : 6 m × 1 m                                                   No. of seedlings damaged by                                                   larvae of Spodoptera litura                                                   Days after spraying                                        Product            2          9                                               ______________________________________                                        1.   Triterpene active fraction                                                                      3.04       2.50                                        2.   Commercial sample 1                                                                             3.27       3.44                                        3.   Commercial sample 2                                                                             3.50       3.08                                        4.   Commercial sample 3                                                                             4.77       5.31                                        5.   Control           8.37       8.91                                        ______________________________________                                        Mustard            Plot size : 6 m × 6 m                                Product            Yield (Quintal/Hectare)                                    ______________________________________                                        1.   Triterpene active fraction                                                                      14.5                                                   2.   Commercial sample 1                                                                             13.7                                                   3.   Commercial sample 4                                                                             11.7                                                   4.   Control           9.5                                                    ______________________________________                                        Cotton             Plot size : 8 m × 8 m                                Percentage bud damage due to spotted bollworm Erias vittella;                 percentage boll damage due to pink bollworm Pectinophora                      gossipiella and seed cotton yield (Quintal/hectare).                          Product      % bud damage                                                                              % boll damage                                                                             Yield                                    ______________________________________                                        1.   Triterpene  32.1        25.4      18.9                                        active fraction                                                          2.   Commercial  32.6        26.5      17.7                                        sample 1                                                                 3.   Commercial  39.7        32.7      14.6                                        sample 3                                                                 4.   Control     45.4        38.5      11.7                                   ______________________________________                                        Safflower          Plot size : 5 m × 5 m                                Percentage of capitula damage due to Helithis armigera and yield              (Quintal/hectare)                                                             Product           % Capitula damage                                                                           Yield                                         ______________________________________                                        1.   Triterpene active fraction                                                                     26.5          15.4                                      2.   Commercial sample 1                                                                            30.4          13.6                                      3.   Commercial sample 4.                                                                           36.5          12.7                                      4.   Control          52.5          11.4                                      ______________________________________                                        Sorghum            Plot size : 6 m × 6 m                                Dead heart percentage due to shootfly Atherigona Soccata, and                 sorghum stem borer Chilo pertellus.                                                                             % Dead                                                   % Dead   % Dead      heart                                                    heart    heart       due to stem                                 Product      21st day after 28 days                                                                             borer.                                      ______________________________________                                        1.   Triterpene  21.8     32.7      10.6                                           active fraction                                                          2.   Commercial  25.4     36.9      14.7                                           sample 1                                                                 3.   Commercial  26.5     37.6      17.6                                           sample 3                                                                 4.   Control     35.6     42.8      38.5                                      ______________________________________                                        Sorghum : GRAIN AND FODDER YIELD                                                               Grain yield Fodder yield                                     Product          Quintal/hectare                                                                           Tonne/hectare                                    ______________________________________                                        1.   Triterepene active                                                                            16.1        8.7                                               fraction                                                                 2.   Commercial sample 1                                                                           14.3        6.9                                          3.   Commercial sample 3                                                                           12.5        6.7                                          4.   Control         8.6         5.4                                          ______________________________________                                    

EXAMPLE 6

Neem seeds which were stored for at least 2-4 months after collectionwere powdered and 10 kg of the powder was stirred with 30 liters ofaqueous (85%) methanol:hexane (1:2) for 2 hours and filtered. Thefiltrate containing two layers one of methanol and other of hexane wereseparated and the solvents removed from both the layers to yield thetriterpenes along with water soluble polar compounds 0.7 kg and 1.8 kgof the lipid fraction respectively.

The lipid fraction (1 kg) was dissolved in acetone (1.5 liters andchilled to 0° to -10° C. for 12 hrs. The solid separated was filtered at0° C. and the filtrate containing the unsaturated lipids wasconcentrated to give the lipid-active fraction, 0.5 kg. The saturatedfats were used for other purposes.

EXAMPLE 7

Neem cake obtained by the expelling oil in an expeller (20 kg) wasstirred with 85% aqueous methanol and hexane (60 liters) (1:2) for threehours and filtered. The aqueous methanol layer and the hexane layer wereseparated and solvent removed separately to give 1.6 kg of mixture oftriterpenes and water soluble polar compounds and 1.6 kg of lipidfraction.

The lipid fraction (1 kg) was dissolved in 1 liter of ethylenedichloride and chilled to 0° to -10° for 12 hrs and the solid separatedwas filtered. The filtrate containing the unsaturated lipids wereconcentrated to give the lipid active fraction. 0.450 kg. The saturatedfats were used for other purposes.

EXAMPLE 8

Neem seeds which were stored atleast for 2-4 months after collectionwere powdered and 10 kg of the powder was stirred with 30 liters ofmethanol:hexane (1:2) for 2 hours and filtered. The filtrate containingtwo layers one of methanol and other of hexane were separated and thesolvents removed from both the layers to yield the triterpenes alongwith water soluble polar compounds 0.7 kg and 1.8 kg of the lipidfraction respectively.

The lipid fraction (1 kg) was dissolved in 1 liter of ethylacetate andchilled at 0° to -10° for 12 hrs. The solid separated was removed byfiltration at 0° and the filtrate which contained unsaturated lipids wasconcentrated to give the lipid active fraction, 0.6 kg. The saturatedfats were used for other purposes.

The bioefficacy data generated during field trials at 40 ppm withlipid-active fraction on different aphids on various crops are given inTable 3 which also shows a comparative bioefficacy with those of someneem pesticides.

                  TABLE 3                                                         ______________________________________                                        BIOEFFICACY DATA OF LIPID-RICH                                                ACTIVE FRACTION DURING FIELD TRIALS                                           AGAINST DIFFERENT APHIDS ON                                                   VARIOUS CROPS.                                                                (Number denotes the average number of apids/5                                 plants).                                                                      ______________________________________                                        Day after  Lipid active                                                       spray      Fraction    Margosan-O  Neemark                                    ______________________________________                                        Precount   48.7        48.2        46.2                                       1st spray                                                                     1          40.0        31.2        40.2                                       2.         31.2        27.7        30.2                                       4.         18.7        27.0        35.2                                       10.        16.2        28.2        37.7                                       14         22.2        30.0        37.0                                       15 second spray                                                               1          17.7        24.7        20.2                                       2          15.0        18.5        25.0                                       4          15.2        27.2        27.7                                       10         16.2        35.7        30.5                                       14         22.2        38.2        37.2                                       15 Third spray                                                                1          17.2        31.2        30.2                                       2          20.2        27.7        40.7                                       4          22.0        29.5        47.0                                       10         22.7        31.7        57.0                                       14         23.7        37.5        57.0                                       15 Fourth spray                                                               1          17.7        22.2        47.2                                       2          20.7        27.5        45.0                                       4          22.5        30.7        48.2                                       10         22.5        30.0        56.2                                       14         22.0        35.0        59.0                                       ______________________________________                                                    Lipid active                                                      Day after   Fraction   Margosan-O  Jawan                                      spray       1       2      1     2     1    2                                 ______________________________________                                        Mustard Aphid Lipaphis erysimi; 1 plot size 6 m × 6 m                   Mustard sawfly: Athalia proxima 2.                                            Precount    38      11     34    13.2  38   11                                First spray                                                                   1           20             22          28                                     2           17             18          26                                     4           15             18          30                                     10          15             18          32                                     14          16             21          37                                     15 Second spray                                                               1           12      7      17    7     30   10                                2           12      7      17    7     27   10                                4           10      7      15    7     29   10                                10          11      8      15    8     31   10                                14          12      8      16    8     34   11                                15 Third spray                                                                1           13      5      10    6     35   9                                 2           13      5      11    6     30   9                                 4           15      6      11    7     32   9                                 10          15      7      11    7     37   9                                 14          17      7      13    7     39   10                                15 Fourth spray                                                               1           12      5      10    6     27   8                                 2           10      5      11    6     25   8                                 4           11      6      12    7     29   9                                 10          11      6      12    7     34   9                                 14          13      6      15    8     39   11                                ______________________________________                                        Safflower Aphid: Daetynotus carthami Plot size 5 m × 5 m                Precount   37          39          37                                         First spray                                                                   1          24          22          30                                         2          21          17          27                                         4          17          17          25                                         10         18          18          21                                         14         18          15          37                                         15 Second spray                                                               1          8           8           28                                         2          6           8           20                                         4          6           9           18                                         10         7           13          19                                         14         6           14          27                                         15 Third spray                                                                1          3           9           20                                         2          3           9           17                                         4          4           9           17                                         10         6           11          22                                         14         6           13          26                                         15 Fourth spray                                                               1          1           7           18                                         2          4           7           17                                         4          4           11          11                                         10         6           14          22                                         14         7           16          26                                         ______________________________________                                        Sorghum Apid : Schizophis Sorshiella Plot size 6 m × 6 m                Day after  Lipid active                                                       spray      Fraction    Margosan-O  Neemark                                    ______________________________________                                        Precount   31.5        36.5        39.5                                       Third spray                                                                   1          16.2        30.2        30.2                                       2          16.0        26.2        27.7                                       4          17.2        24.2        22.5                                       10         18.2        26.7        28.5                                       14         19.2        29.5        29.2                                       15 Fourth spray                                                               1          13.5        21.7        21.5                                       2          14.5        21          17.7                                       5          14.0        23.7        21.5                                       10         15.2        26.3        24.7                                       14         15.5        28.2        29.2                                       ______________________________________                                    

We claim:
 1. Isolated Triterpene derivative of Azadirachtin according to the formula ##STR1## wherein R₁ is Bez, R₂ is Ac, R₃ is OH and R₄ is COOCH₃ ;R₁ is Tig, R₂ is Ac, R₃ is H and R₄ is OH; or R₁ is H, R₂ is Tig, R₃ is COOCH₃ and R₄ is H;where Bez represents benzoyl, Ac represents acetyl and Tig represents tigloyl. 